Naked DNA vaccines have shown promise for stimulating protective immunity by either T helper (Th) 1 cell- or Th2 cell subsets. However, delivery has been primarily limited to peripheral sites rather than mucosal tissues. As a result, minimal secretory (S)-IgA responses or immune mucosal Th cells are induced. To successfully immunize the mucosa, efforts must consider the natural barriers at mucosal surfaces, and effectively target mucosal inductive tissues. In an effort to facilitate optimal mucosal immunity, we propose to utilize a novel approach for DNA delivery whereby the expression plasmid is complexed with an M cell targeting molecule, reovirus protein sigma 1, to ferry the DNA to mucosal inductive tissues following intranasal (i.n.) immunization. Much like live vector vaccine delivery systems which mediate host entry via M cells, we hypothesize that the M cell ligand, protein sigma 1, will direct the DNA to the nasal-associated lymphoid tissue (NALT) for the appropriate stimulation of mucosal B and T cell subsets at local and distal mucosal tissues. To enable this effort, studies in Specific Aim 1 are focused on assessing the types and magnitude of mucosal antibody responses and the supportive CD4+ T cells induced following i.n. immunization with the protein sigma 1-directed DNA vaccine encoding humanized HIV gp140 or gp160. In addition, assessment of neutralizing mucosal and serum anti-HIV (Bal) antibodies will be determined. Studies in Specific Aim 2 are focused on optimizing CTL responses against HIV gp140 or gp160. The efficacy of this CTL activity will be determined by reduction of plaque-forming units of vaccina-gp160 construct subsequent mucosal delivery. These studies will provide the basis for future development of subunit vaccines to target the NALT for eliciting protective immunity via the common mucosal immune system.